The overall goal of the proposed research is to elucidate the function and physiological substrate specificity of protein L-isoaspartyl methyltransferase (PIMT), an enzyme enriched in brain and neuroendocrine tissues. PIMT transfers methyl groups from Sadenosyl-L-methionine onto the free carboxyl group of atypical beta-linked aspartyl residues. Formation of these isopeptide bonds has been shown to be a major source of spontaneous protein damage under physiological conditions. In vitro, PIMT has been shown to catalyze the conversion of the isopeptide linkage back to a normal linkage, adding support to the idea that it may repair damaged proteins in vivo. In exploring this hypothesis further, our first aim will be to characterize a major substrate for PIMT in rat PC12 cells, determine the site of methylation, and determine if, as predicted by the repair hypothesis, this substrate accumulates isoaspartate when PIMT activity is inhibited. Our second aim is to use a PCR-based approach to determine how many distinct isozymes of PIMT exist, to determine how they differ in sequence, and to determine if they are all generated by alternative splicing of a single gene. Our third aim is to characterize a 30 kD protein from cow brain that binds reversibly to the type II isozyme of PIMT. We wish to know how this protein binds to PIMT II, where it is localized in the cell, and if its sequence is similar to any previously characterized protein of known function. Our final aim is to map the PIMT gene to a defined region of a human chromosome. If it maps near any hereditary diseases of unknown etiology, we will assay tissues or cell lines representative of these diseases to determine if a defect in PIMT expression might be the cause. The proposed studies should provide important new information on an enzyme which appears to play a key role in the control of spontaneous damage to cellular proteins.